Study On Screening Of SMO Inhibitors And Their Antitumor Mechanism

Objective Spermine oxidase(SMO)is an amine oxidase that participates in the polyamine catabolism and its abnormal expression is closely related to the occurrence and development of many diseases such as cancer,thus becomes a new molecular target for anti-tumor therapy.The purpose of this study is to design and screen new SMO small molecule inhibitor on the basis of pharmacophore by computer aided drug design technology,and then to analyze its inhibitory effects on SMO activity in the cellular and molecular levels,and to evaluate its effects on the cell proliferation,apoptosis and autophagy using human A549 lung cancer line as a cell model.Methods 1)Computer-aided drug design technique was used to virtual screen potential small molecule inhibitors that targets SMO;2)MTT method was used to assay the proliferation of A549 lung cancer cells;3)Prokaryotic-expressed human SMO recombinant protein was purified by Ni-NTA affinity chromatography;4)Chemiluminescence assay was used to determine SMO activity;5)HPLC was performed to determine the polyamine content in A549 cells;6)the migration ability of A549 cells was evaluated by Transwell method;7)Flow cytometry assay was used to analyze cell cycle and to determine cell ratio of apoptotic cells;8)Western blot assay was used to determine expression of related genes in protein level;9)Electron microscope and confocal microscopy were used to observe autophagy.Results 1)By the computer aided drug design technology,the pharmacophore module was used to establish the pharmacophore model based on the interactions between SMO and its substrate Spm.And then this model was used as query structure to screen the SPECS chemical structures database.By docking the hit compounds with SMO by Autodock software,28 potential SMO small molecule inhibitors were finally obtained;2)MTT analysis found that,among these 28 small molecular compounds,SI-4650 had the strongest inhibitory ability on the proliferation of A549 cells and this compound was then chosen for further analysis;3)Prokaryotic expression and purification of recombinant human SMO which had the enzyme activity was successfully performed.It was found that SI-4650 could significantly inhibit the enzyme activity of this recombinant SMO;4)SI-4650 could interfere with polyamine metabolism,increase Spm but decrease Spd levels,and significantly reduce total polyamine content in A549 cells;5)SI-4650 effectively inhibited the proliferation and migration of A549 cells and induced a cell cycle arrest in S/G2 phases;6)Treating A549 cells with SI-4650 could induce apoptosis,which caused the percentage of apoptotic cells to increase from 4.54% to 17.41%,and also an increased Bax/Bcl-2 ratio;7)Treating A549 cells with SI-4650 could increase the expression of autophagin LC3 B and its transfer to autophagosome,decrease level of the p62(an autophagic substrate)and cause autophagic death in A549 cells.Conclusion This study has successfully designed and screened out a small molecule SMO inhibitor SI-4650 which could: 1)effectively interfere with polyamine metabolism,2)inhibit cell cycle,cell proliferation and cell migration,3)induce apoptosis and autophagic cell death in human A549 lung cancer cells.The above research results will provide a new tool and mean for anti-tumor research based on the polyamine metabolism,and provide a new potential drug for clinical treatment of tumors.